Lettuce named DIP 6992

ABSTRACT

A novel leaf lettuce cultivar, designated DIP 6992, is disclosed. The invention relates to the seeds of lettuce cultivar DIP 6992, to the plants of lettuce cultivar DIP 6992 and to methods for producing a lettuce plant by crossing lettuce cultivar DIP 6992 with itself or another lettuce line. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce lines derived from the cultivar DIP 6992.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive erect, redoakleaf lettuce (Lactuca sativa) cultivar, designated DIP 6992. Allpublications cited in this application are herein incorporated byreference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.In lettuce, the important traits include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, better post-harvest shelf-life of theleaves, better standing ability in the field, better uniformity, andbetter agronomic quality.

Most cultivated forms of lettuce belong to the highly polymorphicspecies Lactuca sativa which is grown for its edible head and leaves. Asa crop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield. Lettuce is theworld's most popular salad. In the United States, the principal growingregions are California and Arizona which produce approximately 329,000acres out of a total annual acreage of more than 333,000 acres (USDA,2005). Fresh lettuce is available in the United States year-roundalthough the greatest supply is from May through October. For plantingpurposes, the lettuce season is typically divided into three categories,early, mid and late, with the coastal areas planting from January toAugust, and the desert regions from August to December. Lettuce isconsumed nearly exclusively as fresh, raw product, and occasionally as acooked vegetable.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositaefamily). Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are several morphological types of lettuce. TheCrisphead group includes the Iceberg and Batavian types. Iceberg lettucehas a large, firm head with a crisp texture and a white or creamy yellowinterior. Batavian lettuce predates Iceberg lettuce and has a smallerand less firm head. The Butterhead group has a small, soft head with analmost oily texture. Romaine lettuce, also known as Cos lettuce, haselongated upright leaves forming a loose, loaf-shaped head and the outerleaves are usually dark green. Leaf lettuce comes in many varieties,none of which form a head. There are three types of lettuce which areseldom seen in the United States: Latin lettuce, which looks like across between Romaine and Butterhead; Stem lettuce, which has long,narrow leaves and thick, edible stems; and Oilseed lettuce, which is aprimitive type of lettuce grown for its large seeds that are pressed toobtain oil.

Lactuca sativa is a simple diploid species with nine pairs ofchromosomes (2N=18). Lettuce is an obligate self-pollinating specieswhich means that pollen is shed before stigma emergence, assuring 100%self-fertilization. Since each lettuce flower is an aggregate of about10-20 individual florets (typical of the Compositae family), manualremoval of the anther tubes containing the pollen is tedious. As aresult, a modified method of misting to wash off the pollen prior tofertilization is needed to assure crossing or hybridization. Flowers tobe used for crossings are selected about 60-90 minutes after sunrise.Selection criteria include plants with open flowers, where the stigmahas emerged and pollen is visibly attached to a single stigma (there areabout 10-20 stigma). Pollen grains are washed off using 3-4 pumps ofwater from a spray bottle and with enough pressure to dislodge thepollen grains without damaging the style. Excess water is then dried offusing clean paper towels and about 30 minutes later, the styles springback up and the two lobes of the stigma are visibly open in a “V” shape.Pollen from another variety or donor parent is then introduced by gentlyrubbing the stigma and style of the donor parent to the maternal parent.Most pertinent information including dates and pedigree are then securedto the flowers using tags.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection andbackcross breeding.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars,nevertheless it is also suitable for the adjustment and selection ofmorphological characters, color characteristics and simply inheritedquantitative characters. Various recurrent selection techniques are usedto improve quantitatively inherited traits controlled by numerous genes.The use of recurrent selection in self-pollinating crops depends on theease of pollination, the frequency of successful hybrids from eachpollination, and the number of hybrid offspring from each successfulcross

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years or more. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a focus on clear objectives.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of lettuce breeding is to develop new, unique and superiorlettuce cultivars. The breeder initially selects and crosses two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. The breeder can theoretically generatebillions of different genetic combinations via crossing, selfing andmutations.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsthat are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.This unpredictability results in the expenditure of large research fundsto develop superior lettuce cultivars.

The development of commercial lettuce cultivars requires the developmentof lettuce varieties, the crossing of these varieties, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits, are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the recurrent parent andthe trait of the donor parent are selected and repeatedly crossed(backcrossed) to the recurrent parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population, will be represented by a progenywhen generation advance is completed

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding John Wiley and Son, pp.115-161, 1960; Allard, 1960; Fehr, 1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Lettuce in general and Leaf lettuce in particular, is an important andvaluable vegetable crop. Thus, a continuing goal of plant breeders is todevelop stable, high yielding lettuce cultivars that are agronomicallysound. The reasons for this goal are obviously to maximize the amount ofyield produced on the land. To accomplish this goal, the lettuce breedermust select and develop lettuce plants that have the traits that resultin superior cultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel red oakleaflettuce cultivar designated DIP 6992. This invention thus relates to theseeds of lettuce cultivar DIP 6992, to the plants or part(s) thereof oflettuce cultivar DIP 6992, to plants or part(s) thereof having all thephenotypic and morphologic characteristics of lettuce cultivar DIP 6992and to plant or part(s) thereof having the phenotypic and morphologiccharacteristics of lettuce cultivar DIP 6992 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions. Parts of the lettuce cultivar of the presentinvention are also provided such as, i.e., pollen obtained from theplant cultivar and an ovule obtained from the plant cultivar.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar DIP 6992. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of lettuce cultivar DIP6992. Preferably, the cells of such tissue culture will be embryos,meristematic cells, seeds, callus, pollen, leaves, anthers, roots, roottips, flowers, stems, and axillary buds. Protoplasts produced from suchtissue culture are also included in the present invention. The lettuceplants regenerated from the tissue culture are also part of theinvention.

Also included in the invention are methods for producing a lettuce plantproduced by crossing lettuce cultivar DIP 6992 with itself or anotherlettuce cultivar. When crossed with itself, that is, when crossed withanother lettuce cultivar DIP 6992 plant or self-pollinated, lettucecultivar DIP 6992 will be conserved. When crossed with another,different lettuce plant, an F₁ hybrid seed is produced. F₁ hybrid seedsand plants produced by growing said hybrid seeds are included in thepresent invention. A method for producing an F₁ hybrid lettuce seedcomprising crossing a lettuce cultivar DIP 6992 plant with a differentlettuce plant and harvesting the resultant hybrid lettuce seed are alsopart of the invention. The hybrid lettuce seed produced by the methodcomprising crossing a lettuce cultivar DIP 6992 plant with a differentlettuce plant and harvesting the resultant hybrid lettuce seed, areincluded in the invention, as are the hybrid lettuce plant or part(s)thereof, and seeds produced by growing said hybrid lettuce seed.

In another aspect, the present invention provides transformed DIP 6992lettuce cultivar plants or part(s) thereof that have been transformed sothat its genetic material contains one or more transgenes, preferablyoperably linked to one or more regulatory elements. Also, the inventionprovides methods for producing a lettuce plant containing in its geneticmaterial one or more transgenes, preferably operably linked to one ormore regulatory elements, by crossing transformed DIP 6992 lettucecultivar plants with either a second plant of another lettuce cultivar,or a non transformed DIP 6992 lettuce cultivar, so that the geneticmaterial of the progeny that results from the cross contains thetransgene(s), preferably operably linked to one or more regulatoryelements. The invention also provides methods for producing a lettuceplant that contains in its genetic material one or more transgene(s),wherein the method comprises crossing the cultivar DIP 6992 with asecond lettuce cultivar of another lettuce cultivar which contains oneor more transgene(s) operably linked to one or more regulatoryelement(s) so that the genetic material of the progeny that results fromthe cross contains the transgene(s) operably linked to one or moreregulatory element(s). Transgenic lettuce cultivars, or part(s) thereofproduced by the methods are in the scope of the present invention.

More specifically, the invention comprises methods for producing a malesterile lettuce plant, an herbicide resistant lettuce plant, an insectresistant lettuce plant, a disease resistant lettuce plant, a waterstress tolerant lettuce plant, a heat stress tolerant lettuce plant, anda lettuce plant with improved shelf-life and delayed senescence. Saidmethods comprise transforming a lettuce cultivar DIP 6992 plant with anucleic acid molecule that confers male sterility, herbicide resistance,insect resistance, disease resistance, water stress tolerance, heatstress tolerance, or improved shelf life and delayed senescence,respectively. The transformed lettuce plants, or part(s) thereof,obtained from the provided methods, including a male sterile lettuceplant, an herbicide resistant lettuce plant, an insect resistant lettuceplant, a disease resistant lettuce plant, a lettuce plant tolerant towater stress, a lettuce plant tolerant to heat stress, a lettuce plantwith improved shelf-life or a lettuce plant with delayed senescence areincluded in the present invention. For the present invention and theskilled artisan, disease is understood to be fungal diseases, viraldiseases, bacterial diseases or other plant pathogenic diseases and adisease resistant plant will encompass a plant resistant to fungal,viral, bacterial and other plant pathogens.

In another aspect, the present invention provides for methods ofintroducing one or more desired trait(s) into lettuce cultivar DIP 6992and plants obtained from such methods. The desired trait(s) may be, butnot exclusively, a single gene, preferably a dominant but also arecessive allele. Preferably, the transferred gene or genes will confersuch traits as male sterility, herbicide resistance, insect resistance,resistance to bacterial, fungal, or viral disease, increased leafnumber, improved shelf-life, delayed senescence and tolerance to waterstress or heat stress. The gene or genes may be naturally occurringgene(s) or transgene(s) introduced through genetic engineeringtechniques. The method for introducing the desired trait(s) ispreferably a backcrossing process making use of a series of backcrossesto lettuce cultivar DIP 6992 during which the desired trait(s) ismaintained by selection.

When using a transgene, the trait is generally not incorporated intoeach newly developed line/cultivar such as DIP 6992 by directtransformation. Rather, the more typical method used by breeders ofordinary skill in the art to incorporate the transgene is to take a linealready carrying the transgene and to use such line as a donor line totransfer the transgene into the newly developed line. The same wouldapply for a naturally occurring trait. The backcross breeding processcomprises the following steps: (a) crossing lettuce cultivar DIP 6992plants with plants of another cultivar that comprise the desiredtrait(s), (b) selecting the F₁ progeny plants that have the desiredtrait(s); (c) crossing the selected F₁ progeny plants with lettucecultivar DIP 6992 plants to produce backcross progeny plants; (d)selecting for backcross progeny plants that have the desired trait(s)and physiological and morphological characteristics of lettuce cultivarDIP 6992 to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) one, two, three, four, five six, seven, eight, nine ormore times in succession to produce selected, second, third, fourth,fifth, sixth, seventh, eighth, ninth or higher backcross progeny plantsthat comprise the desired trait(s) and the physiological andmorphological characteristics of lettuce cultivar DIP 6992 as determinedin Table 1 at a 5% significance level when grown in the sameenvironmental conditions. The lettuce plants produced by the methods arealso part of the invention. Backcrossing breeding methods, well-knownfor a man skilled in the art of plant breeding, will be furtherdeveloped in subsequent parts of the specification.

In a preferred embodiment, the present invention provides methods forincreasing and producing lettuce cultivar DIP 6992 seed, whether bycrossing a first parent lettuce cultivar plant with a second parentlettuce cultivar plant and harvesting the resultant lettuce seed,wherein both said first and second parent lettuce cultivar plant are thelettuce cultivar DIP 6992 or by planting a lettuce seed of the lettucecultivar DIP 6992, growing a lettuce cultivar DIP 6992 plant from saidseed, controlling a self-pollination of the plant where the pollenproduced by a grown lettuce cultivar DIP 6992 plant pollinates theovules produced by the very same lettuce cultivar DIP 6992 grown plantand harvesting the resultant seed.

The invention further provides methods for developing lettuce cultivarsin a lettuce breeding program using plant breeding technique includingrecurrent selection, backcrossing, pedigree breeding, molecular markers(Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs). AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, etc.) enhancedselection, genetic marker enhanced selection and transformation. Seeds,lettuce plants, and part(s) thereof produced by such breeding methodsare also part of the invention.

Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a gene, allof which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Maturity Date. Maturity refers to the stage when plants are of full sizeor optimum weight, and in marketable form or shape to be of commercialor economic value. In leaf types they range from 50-75 days from time ofseeding, depending upon the season of the year.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort Society Enterprise Ltd RHS Garden; Wisley,Woking; Surrey GU236QB, UK.

Lettuce Yield (Tons/Acre). The yield in tons/acre is the actual yield ofthe lettuce at harvest.

Plant Part. As used herein, the term “plant part” includes leaves,stems, roots, seed, embryos, pollen, ovules, flowers, root tips,anthers, tissue, cells, axillary buds, and the like.

Plant Cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering wherein essentially allthe desired morphological and physiological characteristics of a varietyare recovered in addition to the single gene transferred into thevariety via the backcrossing technique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar DIP 6992 has superior characteristics and was developedfrom a cross designated 20839 which corresponds to a cross between228120/3×228106/4, made in the summer of 2003 in a greenhouse. The F₁plants were grown in a greenhouse during the winter of 2003/2004. The F₂20839/02 was tested on Bremia races B120, B121 and B123 in spring 2004in a Vilmorin pathology lab in France. The F₂ 20839/02 was also testedfor Nasonovia resistance in summer 2004 in a Vilmorin pathology lab inFrance. The F₂ 20839/02 was sown in the summer 2004 in France anddesignated sowing number 4/13701. Five plants were chosen, 4/13701 /01to /05, and grown in a glasshouse during fall 2004 in France. The F₃genotype 4/13701/04 was tested on Bremia races B120, B121, B123 and forNasonovia resistance in La Ménitré, France, showing resistance to Bremiaraces and segregating for Nasonovia resistance. This F₃ genotype wassown in summer 2005 under a sowing number designated 5/14633, where 13plants were selected from the plot and grown under a glasshouse inFrance in fall 2005. The F₄ genotype 5/14633/13 was tested on Bremiaraces B124 and B125 and Nasonovia resistance during winter 2005/2006 inLa Ménitré, France and showing resistance to Bremia and Nasonovia.Plants were sown in summer 2006 under a sowing number designated 6/6992in France and 6 plants were selected from the plot and grown under aglasshouse in fall 2006 in La Ménitré, France. The F₅ genotype 6/6992/03was controlled for Bremia and Nasonovia resistance. A small F₆ seed lotwas produced in La Ménitré, France and a larger seed lot in 2008 at thesame location. This F₇ seed lot was controlled for Bremia, Nasonovia anduniformity in the field during fall 2008.

Cultivar DIP 6992 is an erect, red oakleaf type similar to lettucecultivar Nougatine but there are numerous differences: lettuce cultivarDIP 6992 has a more erect shape and has more fresh red-colored leaves.Lettuce cultivar DIP 6992 has thinner and narrower leaves than lettucecultivar Nougatine. Additionally, lettuce cultivar DIP 6992 has a fastergrowth habit than lettuce cultivar Nougatine, which provides a shorterproduction cycle. Lettuce cultivar DIP 6992 has Nasonovia resistance,while lettuce cultivar Nougatine does not have Nasonovia resistance.

Lettuce cultivar DIP 6992 is a red oakleaf leaf lettuce with anoriginal, attractive, spiky leaf shape and erect shape. The erect shapeof lettuce cultivar DIP 6992 is highly adapted to the “Baby leaf”lettuce production. Lettuce cultivar DIP 6992 has shown goodadaptability to the Salinas, Calif. area. Lettuce cultivar DIP 6992 isresistant to all European official Bremia races BI1 to BI26.

Some of the criteria used to select various generations include: color,disease resistances, head weight, number of leaves, leaf appearance,strength and length, yield, taste (not bitter), process ability,emergence, maturity, plant architecture, seed yield and quality.

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits and has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. Lettuce cultivar 6992 has beenincreased with continued observation for uniformity. No variant traitshave been observed or are expected in lettuce cultivar DIP 6992.

Lettuce cultivar DIP 6992 looks like a curly endive but belongs to thesativa species.

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant: Type: Leafy, non-headingSeed: Color: Black Cotyledon to Fourth Leaf Stage: AnthocyaninDistribution: Present Mature Leaves: Green color: Deep Red AnthocyaninDistribution: Present Length: 30-35 cm Glossiness: Strong Blistering:None Leaf Thickness: Medium Plant (at Market Stage): Plant Shape: Veryerect Plant Weight: 300-600 grams Plant Firmness: Firm Core: Diameter atBase of Head: 10 mm Maturity: Summer: 4 days earlier than NougatineWinter: 8 days earlier than Nougatine Adaptation: Primary Regions ofAdaptation (tested and proven adapted): Southwest (California, Arizonadesert) Season: Spring, summer, and fall Soil Type: Adapted to most soiltypes Diseases: Fungal/Bacterial: Downy Mildew (Bremia lactucae): Verystrong resistance to Bl 1-10, 11-18, 20-26 Sclerotinia Rot: Strongresistance Nasonovia ribisnigri: Resistant (Nougatine is susceptible)Physiological/Stress: Tipburn: Strong resistance

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a lettucecultivar plant by crossing a first parent lettuce cultivar plant with asecond parent lettuce cultivar plant, wherein either the first or secondparent lettuce cultivar plant is a lettuce cultivar DIP 6992 plant.Further, both first and second parent lettuce plants can come fromlettuce cultivar DIP 6992. When self-pollinated, or crossed with anotherlettuce cultivar DIP 6992 plant, lettuce cultivar DIP 6992 will bestable, while when crossed with another, different lettuce cultivarplant, an F₁ hybrid seed is produced.

Still further, this invention is also directed to methods for producinga DIP 6992-derived lettuce plant by crossing lettuce cultivar DIP 6992with a second lettuce plant and growing the progeny seed, and repeatingthe crossing and growing steps with the DIP 6992-derived plant from 0 to7 times. Thus, any such methods using the lettuce cultivar DIP 6992 arepart of this invention: selfing, backcrosses, hybrid production, crossesto populations, and the like. All plants produced using lettuce cultivarDIP 6992 as a parent are within the scope of this invention, includingplants derived from cultivar DIP 6992. Advantageously, lettuce cultivarDIP 6992 is used in crosses with other, different, cultivars to producefirst generation (F₁) lettuce seeds and plants with superiorcharacteristics.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, roots, root tips, leaves, meristematic cells, axillary buds,stems, anthers, and the like.

As is well-known in the art, tissue culture of lettuce can be used forthe in vitro regeneration of a lettuce plant. Tissue culture of varioustissues of lettuce and regeneration of plants therefrom is well knownand widely published. For example, reference may be had to Teng et al.,HortScience. 1992, 27: 9, 1030-1032 Teng et al., HortScience. 1993, 28:6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46:3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38:1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279,1441-1449, Nagata et al., Journal for the American Society forHorticultural Science. 2000, 125: 6, 669-672. Thus, it is clear from theliterature that the state of the art is such that these methods ofobtaining plants are “conventional” in the sense that they are routinelyused and have a very high rate of success. Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce lettuce plants having the physiological and morphologicalcharacteristics of lettuce cultivar DIP 6992.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed cultivar.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedlettuce plants, using transformation methods as described below toincorporate transgenes into the genetic material of the lettuceplant(s).

Expression Vectors for Lettuce Transformation—Marker Genes:

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well-known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include beta-glucuronidase (GUS,beta-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), De Block et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells. Chalfieet al., Science 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Lettuce Transformation—Promoters:

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well-known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners, Gatzet al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10Gatz et al., Mol. Gen. Genetics 227:229-237 (1991). A particularlypreferred inducible promoter is a promoter that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter is the inducible promoter from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inlettuce or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin lettuce. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in lettuce. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993).

Signal Seguences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Nat. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Stiefel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which arewell-understood in the art, yield a plurality of transgenic plants whichare harvested in a conventional manner, and a foreign protein then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is lettuce. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology, CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btdelta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT application US93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor 1), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Pratt et al., Biochem. Biophys. Res. Comm. 163:1243(1989) (an allostatin is identified in Diploptera puntata). See alsoU.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo alpha-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-alpha-1,4-D-galacturonase. See Lamb et al., BioTechnology10:1436 (1992). The cloning and characterization of a gene which encodesa lettuce endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

S. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

2. Genes that Confer Resistance to a Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy proprionic acids and cyclohexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSPS which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Seealso Mohapatra in Transgenic Research. 1999, 8: 1, 33-44 that disclosesLactuca sativa resistant to glufosinate. European patent application No.0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman etal., disclose nucleotide sequences of glutamine synthetase genes whichconfer resistance to herbicides such as L-phosphinothricin. Thenucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.,DeGreef et al., BioTechnology 7:61 (1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy proprionic acids and cyclohexones, suchas sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait. Such as:

A. Increased iron content of the lettuce, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae. 2000, 521, 101-109. Parallel to the improved iron contentenhanced growth of transgenic lettuce was also observed in earlydevelopment stages.

B. Decreased nitrate content of leaves, for example by transforming alettuce plant with a gene coding for a nitrate reductase. See forexample Curtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

C. Increased sweetness of the lettuce by transferring a gene coding formonellin, that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia et al., Biotechnology. 1992, 10: 5, 561-564.

D. Delayed senescence or browning by transferring a gene or acting onthe transcription of a gene involved in the plant senescence. See Wanget al. In Plant Mol. Bio. 52:1223-1235 (2003) on the role of thedeoxyhypusine synthase in the senescence. See also U.S. Pat. No.6,538,182 issued Mar. 25, 2003.

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Agrobacterium-mediated transformation success has been achieved in riceand corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat.No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 microns. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al. PI. Cell. Rep. 12(3, January), 165-169 (1993), Aragao, F. J. L.,et al. Plant Mol. Biol. 20(2, October), 357-359 (1992), Aragao, F. J.L., et al. PI. Cell. Rep. 12(9, July), 483-490 (1993). Aragao Theor.Appl. Genet. 93: 142-150 (1996), Kim, J.; Minamikawa, T. Plant Science117: 131-138 (1996), Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,BioTechnology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., BioTechnology 10:268 (1992)

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., BioTechnology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994). See also Chupean et al.,BioTechnology. 1989, 7: 5, 503-508.

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic cultivar. The transgenic cultivar could then becrossed, with another (non-transformed or transformed) cultivar, inorder to produce a new transgenic lettuce cultivar. Alternatively, agenetic trait which has been engineered into a particular lettucecultivar using the foregoing transformation techniques could be movedinto another cultivar using traditional backcrossing techniques that arewell known in the plant breeding arts. For example, a backcrossingapproach could be used to move an engineered trait from a public,non-elite cultivar into an elite cultivar, or from a cultivar containinga foreign gene in its genome into a cultivar or cultivars which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

Single Gene Conversions

When the term lettuce plant, cultivar or lettuce line are used in thecontext of the present invention, this also includes any cultivar whereone or more desired traits has been introduced through backcrossingmethods, whether such trait is a naturally occurring one or a transgenicone. Backcrossing methods can be used with the present invention toimprove or introduce a characteristic into the cultivar. The term“backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing 1, 2, 3,4, 5, 6, 7, 8, 9 or more times to the recurrent parent. The parentallettuce plant which contributes the gene or the genes for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental lettuce plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol.

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second cultivar (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a lettuce plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, generally determined at a 5% significance levelwhen grown in the same environmental conditions, in addition to the geneor genes transferred from the nonrecurrent parent. It has to be notedthat some, one, two, three or more, self pollination and growing ofpopulation might be included between two successive backcrosses. Indeed,an appropriate selection in the population produced by the selfpollination, i.e. selection for the desired trait and physiological andmorphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non limiting example of such a protocolwould be the following: a) the first generation F₁ produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and physiological and morphological characteristics of parent A. Stepc) may or may not be repeated and included between the backcrosses ofstep d.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalcultivar. To accomplish this, a gene or genes of the recurrent cultivaris modified or substituted with the desired gene or genes from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original cultivar. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing but also follow up of the characteristic(s) through geneticallyassociated markers and molecular assisted breeding tools. For example,selection of progeny containing the transferred trait is done by directselection, visual inspection for a trait associated with a dominantallele, while the selection of progeny for a trait that is transferredvia a recessive allele require selfing the progeny to determine whichplant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance. An example of a gene controlling resistanceto the lettuce leaf aphid Nasonovia ribisnigri (Nr gene) can be found inVan der Arend and Schijndel in Breeding for Resistance to insects andMites, IOBC wprs Bulletin 22(10), 35-43 (1999). These genes aregenerally inherited through the nucleus. Several of these single genetraits are described in U.S. Pat. Nos. 5,777,196; 5,948,957 and5,969,212, the disclosures of which are specifically hereby incorporatedby reference.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred corn line development in the United States,according to Hallauer, A. R. et al. (1988) “Corn Breeding” Corn and CornImprovement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc, “Principles of Plant Breeding”).The method makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a variety with exactly the adaptation, yielding ability andquality characteristics of the recurrent parent but superior to thatparent in the particular characteristic(s) for which the improvementprogram was undertaken. Therefore, this method provides the plantbreeder with a high degree of genetic control of his work.

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are for example, the transfer of stem rustresistance from “Hope” wheat to “Bart” wheat and even pursuing thebackcrosses with the transfer of bunt resistance to create “Bart 38”,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in “CaliforniaCommon” alfalfa to create “Caliverde”. This new “Caliverde” varietyproduced through the backcross process is indistinguishable from“California Common” except for its resistance to the three nameddiseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,colour characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, “Calady”, has been produced by Jones andDavis. As dealing with quantitative characteristics, Jones and Davisselected the donor parent with the view of sacrificing some of theintensity of the character for which it was chosen, i.e. grain size.“Lady Wright”, a long-grain variety was used as the donor parent and“Coloro”, a short-grain, was used as the recurrent parent. After fourbackcrosses, the medium-grain type variety “Calady” was produced.

Tables

As shown in Table 2 below, lettuce cultivar DIP 6992 is compared tocommercial cultivar Nougatine and commercial cultivar Amboni for leafcolor and plant shape, for which there are significant differences fromthe comparison varieties. Data were taken in 2006 in La Ménitré, France.Column one shows the variety name, column two shows the leaf color andcolumn three shows the plant shape.

TABLE 2 Characteristic Variety Leaf color Plant shape DIP 6992 Mediumbright-red Very erect Nougatine Dark brown-red Medium erect Amboni Darkbright-red Slightly erect

As shown in Table 3 below, lettuce cultivar DIP 6992 is compared tolettuce cultivars Nougatine and Amboni for Bremia and Nasonoviaresistance for which there are significant differences from thecomparison varieties. Column one shows the variety name, column twoshows the Bremia resistance and column three shows the Nasonoviaresistance

TABLE 3 Characteristic Variety Bremia Nasonovia DIP 6992 Resistant toBl24 Resistant Nougatine Resistant to Bl24 Susceptible AmboniSusceptible to Bl24 Susceptible

Deposit Information

A deposit of the Vilmorin SA, Route du Manoir, 49250, La Ménitré, Franceproprietary lettuce cultivar DIP 6992 disclosed above and recited in theappended claims has been made under the Budapest Treaty with NationalCollections of Industrial, Food and Marine Bacteria (NCIMB), 23 StMachar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom. The date ofdeposit was Mar. 10, 2009. The deposit of 2,500 seeds was taken from thesame deposit maintained by Vilmorin SA since prior to the filing date ofthis application. Access to this deposit will be available during thependency of this application to persons determined by the Commissionerof Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14and 35 USC §122. All restrictions upon the deposit will be removed upongranting of a patent, and the deposit is intended to meet all of therequirements of 37 C.F.R. §§1.801-1.809. The NCIMB accession number isNCIMB No. 41616. The deposit will be maintained in the depository for aperiod of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedas necessary during that period.

Although the foregoing invention has been described in some detail byway of example for purposes of clarity and understanding, it will beobvious that certain changes and modifications such as single genemodifications and mutations, somaclonal variants, variant individualsselected from large populations of the plants of the instant line andthe like may be practiced within the scope of the invention, as limitedonly by the scope of the appended claims.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafter areinterpreted to include all such modifications, permutations, andsub-combinations as are within their true spirit and scope.

1. A seed of lettuce cultivar designated DIP 6992, wherein arepresentative sample of seed of said cultivar has been deposited underNCIMB No.
 41616. 2. A lettuce plant, or a part thereof, produced bygrowing the seed of claim
 1. 3. A lettuce plant or a part thereof,having the physiological and morphological characteristics of lettucecultivar DIP 6992 listed in Table
 1. 4. A lettuce plant, or a partthereof, having the physiological and morphological characteristics oflettuce cultivar DIP 6992, wherein a representative sample of seed ofsaid cultivar has been deposited under NCIMB No.
 41616. 5. A tissueculture of regenerable cells produced from the plant of claim 2, whereinsaid cells of the tissue culture are produced from a plant part selectedfrom the group consisting of embryos, meristematic cells, leaves,pollen, root, root tips, stems, anther, axillary buds, flowers or seeds.6. A lettuce plant regenerated from the tissue culture of claim 5, saidplant having the morphological and physiological characteristics oflettuce cultivar DIP 6992, wherein a representative sample of seed hasbeen deposited under NCIMB No.
 41616. 7. A method for producing alettuce seed comprising crossing a first parent lettuce plant with asecond parent lettuce plant and harvesting the resultant hybrid lettuceseed, wherein said first or second parent lettuce plant is the lettuceplant of claim
 2. 8. A hybrid lettuce seed produced by the method ofclaim
 7. 9. A method for producing an herbicide resistant lettuce plantcomprising transforming the lettuce plant of claim 2 with a transgenethat confers herbicide resistance to an herbicide selected from thegroup consisting of imidazolinone, sulfonylurea, glyphosate,glufosinate, L-phosphinothricin, triazine, and benzonitrile.
 10. Anherbicide resistant lettuce plant, or a part thereof, produced by themethod of claim
 9. 11. A method for producing an insect resistantlettuce plant comprising transforming the lettuce plant of claim 2 witha transgene that confers insect resistance.
 12. An insect resistantlettuce plant, or a part thereof, produced by the method of claim 11.13. A method for producing a disease resistant lettuce plant comprisingtransforming the lettuce plant of claim 2 with a transgene that confersdisease resistance.
 14. A disease resistant lettuce plant, or a partthereof, produced by the method of claim
 13. 15. A method for producinga delayed senescence lettuce plant comprising transforming the lettuceplant of claim 2 with a transgene that confers delayed senescence.
 16. Adelayed senescence lettuce plant, or a part thereof, produced by themethod of claim
 15. 17. A method for producing a male sterile lettuceplant comprising transforming the lettuce plant of claim 2 with atransgene that confers male sterility.
 18. A male sterile lettuce plant,or a part thereof, produced by the method of claim
 17. 19. A method ofintroducing a desired trait into lettuce cultivar DIP 6992 comprising:(a) crossing a lettuce cultivar DIP 6992 plant grown from lettucecultivar DIP 6992 seed, wherein a representative sample of seed has beendeposited under NCIMB No. 41616, with plants of another lettuce cultivarplant that comprise a desired trait to produce F₁ progeny plants,wherein the desired trait is selected from the group consisting of malesterility, herbicide resistance, insect resistance, disease resistance,water stress tolerance, heat tolerance, improved shelf life and delayedsenescence; (b) selecting one or more progeny plants that have thedesired trait to produce selected progeny plants; (c) crossing theselected progeny plants with the lettuce cultivar DIP 6992 plants toproduce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait and physiological and morphologicalcharacteristics of lettuce cultivar DIP 6992 listed in Table 1 toproduce selected backcross progeny plants; and (e) repeating steps (c)and (d) ene three or more times in succession to produce selected secondor higher backcross progeny plants that comprise the desired trait andthe physiological and morphological characteristics of lettuce cultivarDIP 6992 listed in Table
 1. 20. A lettuce plant produced by the methodof claim 19, wherein the plant has the desired trait and thephysiological and morphological characteristics of lettuce cultivar DIP6992 listed in Table
 1. 21. A method for producing lettuce cultivar DIP6992 seed, wherein a representative sample of seed has been depositedunder NCIMB No. 41616, comprising crossing a first parent lettucecultivar with a second parent lettuce cultivar and harvesting theresultant lettuce seed, wherein both said first and second lettucecultivars are the lettuce cultivar of claim 4.